The innate immune system is required to perform two difficult tasks: (1) sense the presence of microbes (2) decide, given the nature of the threat, whether and how to mount an immune response. To achieve these tasks, the innate immune system preferentially responds to molecular signatures of microbes that are unique to microbes, that are widely conserved among microbes, and that are difficult for microbes to alter. There is currently great interest in determining what molecular features of pathogens are sensed, and how these features are sensed by the innate immune system. By understanding the fundamental principles of how immune responses are stimulated, it is expected that more effective vaccines and immune therapeutics can be created. In this application, data is presented that mammalian host cells can detect the cytosolic presence of a unique nucleic acid that is produced by most species of bacteria, but is not produced by eukaryotic cells. The molecule in question is a cyclic dinucleotide called cyclic-di-GMP (c-di-GMP). C-di-GMP plays central roles in bacterial physiology, functioning as an critical second messenger in many bacterial pathogens. Our overall hypothesis is that the innate immune system has developed a novel mechanism to sense the presence of cytosolic c-di-GMP, a bacterial second messenger that is not found in eukaryotes. Our preliminary data demonstrate that c-di-GMP is sensed cytosolically by a signaling pathway leading to the potent induction of type I interferons (IFNs) and other co-regulated genes. The potency of c-di-GMP, and the pattern of host gene expression it induces, appears to be comparable to that induced by cytosolic delivery of DNA. We show that induction of innate immune signaling pathways by c-di-GMP requires the TBK1 kinase and its target, the IRF3 transcription factor. By contrast, macrophages deficient in Toll-like receptor -/- -/- (TLR) signaling (MyD88 Trif ) still respond normally to c-di-GMP, as do macrophages deficient in cytosolic -/- RNA sensing (Mavs ). Thus, the host sensor that detects cyclic-di-GMP appears to be novel. Interestingly, our preliminary data suggest that a putative DNA sensor, DAI, may play a role in cytosolic sensing of cyclic-di- GMP. Characterizing the molecular basis by which host cells sense c-di-GMP will contribute an important facet to our understanding of how immune responses are triggered by bacterial pathogens. Thus, our Specific Aims are (1) to identify the components of the host signaling pathway that senses and responds to c-di-GMP leading to the expression of type I interferons and other innate immune response genes;and (2) to test the hypothesis that DAI functions as a mammalian c-di-GMP sensor. Public Health Relevance: Adjuvants are a critical component of safe and effective vaccines. Adjuvants function by stimulating cells of the innate immune system. A better understanding of how adjuvants stimulate innate immunity will allow for the design of more effective and/or safer adjuvants.